Exploring chondrocyte integrin regulation of growth factor IGF-I expression from a transient pAAV vector.

摘要

Insulin-like Growth Factor I (IGF-I) is a growth factor that stimulates both mitogenic and anabolic responses in articular chondrocytes. While it has been shown that exogenous IGF-I can regulate chondrocyte integrins, little is known regarding regulatory effects of IGF-I produced from a transiently expressed plasmid based adeno-associated virus (pAAV) vector. Because chondrocytes are using cellular machinery to overexpress IGF-I, it is of interest to see whether or not pAAV IGF-I will significantly upregulate or downregulate chondrocyte integrins. Additionally, it is of interest to know whether chondrocyte adhesion through integrins will have any regulatory effects on the production of IGF-I from the transgene. Therefore, this study will ascertain if pAAV IGF-I will have similar effects that exogenous IGF-I has on integrin regulation and if integrin silencing mechanisms will affect the production of IGF-I from the transgene.;To test these hypotheses, adult articular chondrocytes were doubly transfected with the pAAV vector for IGF-I and short interference ribonucleic acid (siRNA) for integrins beta1 and alphaV. Gene products were monitored at the transcriptional levels using quantitative real time polymerase chain reactions (qPCR) and IGF-I protein production was monitored at the translational level using enzyme linked immunoabsorbant assays (ELISAs). Adult articular chondrocytes doubly transfected were encapsulated in a three dimensional hydrogel system to simulate an in vivo environment. Samples were collected for analysis at days 2, 4, and 6 post encapsulation. Results show that IGF-I treatment with the pAAV vector does not cause significant changes in the transcriptional regulation of the beta1 integrin in a three dimensional hydrogel system. The pAAV IGF-I vector did not cause significant regulatory changes on integrin alphaV at any time point during the experiment. Additionally, by knocking down the expression levels of integrins by using siRNA, it was shown that integrin knockdown does not have a significant regulatory effect on transcriptional or translational expression levels of IGF-I from the pAAV vector.